Objective     To construct eukaryotic expression vector of siRNA for human cubilin gene and detect its specific interference in human renal proximal tubular epithelial cell line (HKC).      Methods     Two eukaryotic expression vectors of siRNA were constructed for human cubilin gene. HKCs were stably transfected with the pSUPER EGFP vector and the two recombinant vectors. Normal HKCs and transfected HKCs were stimulated by human serum albumin (HSA). Then cubilin mRNA expression of HKCs were detected by fluorescence-based quantitative RT-PCR (FQ-PCR). Endocytosis of TRITC labeled bovine serum albumin (TRITC-BSA) by HKCs was detected by laser scanning confocal fluorescence microscope.      Results     DNA sequencing showed that the target segments were cloned into pSUPER EGFP vector respectively. The results of FQ-PCR indicated there was dose-dependent cubilin mRNA expression in HKCs stimulated with HSA and both vectors could inhibit expression of cubilin gene. The expression of cubilin obviously decreased by 66% and 37% respectively in the transfected HKCs with the two recombinant vectors. TRITC-BSA uptake was obviously lower in HKCs transfected with two recombinant vectors.      Conclusion     The eukaryotic expression vector of siRNA for human cubilin gene was successfully constructed. The expression of cubilin gene and albumin uptake were inhibited effectively by the expressed siRNA.